Immunoglobulin Fc polypeptides

Abstract

Methods and compositions involving polypeptides having an aglycosylated antibody Fc domain. In certain embodiments, polypeptides have an aglycosylated Fc domain that contains one or more substitutions compared to a native Fc domain. Additionally, some embodiments involve an Fc domain that is binds some Fc receptors but not others. For example, polypeptides are provided with an aglycosylated Fe domain that selectively binds FcγRI at a level within 2-fold of a glycosylated Fc domain, but that is significantly reduced for binding to other Fc receptors. Furthermore, methods and compositions are provided for promoting antibody-dependent cell-mediated toxicity (ADCC) using a polypeptide having a modified aglycosylated Fc domain and a second non-Fc binding domain, which can be an antigen binding region of an antibody or a non-antigen binding region. Some embodiments concern antibodies with such polypeptides, which may have the same or different non-Fc binding domain.

Description

[0001]This application claims the priority benefit of U.S. Application No. 61 / 221,999, filed Jun. 30, 2009. Furthermore, the entire disclosures of U.S. Application No. 60 / 915,183 filed on May 1, 2007 and U.S. Application No. 60 / 982,652 filed on Oct. 25, 2007, are specifically incorporated herein by reference in their entirety without disclaimer.BACKGROUND OF THE INVENTION[0002]1. Field of the Invention[0003]The present invention relates generally to the field of protein engineering. More particularly, it concerns improved methods and compositions for the screening of combinatorial antibody Fc libraries expressed in bacteria.[0004]2. Description of Related Art[0005]Currently recombinant therapeutic antibodies have sales of well over $10 bn / yr and with a forecast of annual growth rate of 20.9%, they are projected to increase to $25 bn / yr by 2010. Monoclonal antibodies (mAbs) comprise the majority of recombinant proteins currently in the clinic, with more than 150 products in studies s...

AI Technical Summary

Benefits of technology

[0015]In some embodiments, there are compositions involving a polypeptide that has an aglycosylated Fc domain from an antibody (“antibody Fc domain”). In additional embodiments, the aglycosylated Fc domain is a variant of a wild-type Fc domain such that the variation allows the Fc domain to specifically bind to one or more Fc receptors. In some embodiments, a polypeptide with an aglycosylated Fc domain variant is able to bind only a subset of Fc receptors that a polypeptide with glycosylated version of the wild-type Fc domain (“glycosylated wild-type Fc domain”) can bind. In specific embodiments, the polypeptide with an aglycosylated Fc domain variant can specifically bind FcγRI; in some cases, it has the affinity or binding ability that is within 2-fold of a polypeptide having a glycosylated wild-type Fc domain. In other embodiments, additionally or alternatively, the polypeptide with an aglycosylated Fc domain variant has significantly reduced affinity or binding ability (50-fold or greater reduction) compared to a polypeptide having a glycosylated wild-type Fc domain. In certain embodiments, the polypeptide with an aglycosylated Fc domain variant has a significantly reduced affinity to or ability to bind FcγRIIb. It is contemplated that a polypeptide may have an affinity or binding ability for FcγRI that is comparable (within 2-fold), as well as significantly reduced affinity or binding ability for FcγRIIB, both as compared to a polypeptide having a glycosylated wild-type Fc domain.

Problems solved by technology

Therapeutic murine mAbs entered clinical study in the early 1980s; however, problems with lack of efficacy and rapid clearance due to patients' production of human anti-mouse antibodies (HAMA) became apparent.

These issues, as well as the time and cost consuming related to the technology became driving forces for the evolution of mAb production technology.

In contrast, aglycosylated antibodies produced in bacteria cannot bind to FcRs and therefore are unable to elicit ADCC.

Thus, while such antibodies are more selective than the molecules naturally produced by the immune system they can nonetheless still mediate undesirable immunological responses.

The use of antibody fragments rather than intact or full length IgGs, in the construction and screening of libraries has been dictated by limitations related to the expression of the much larger IgGs in microorganisms.

E. coli possesses a reducing cytoplasm that is unsuitable for the folding of proteins with disulfide bonds which accumulate in an unfolded or incorrectly folded state (Baneyx and Mujacic, 2004).

However, because antibody fragments lack the Fc domain, they do not bind the FcRn receptor and are cleared quickly; thus, they are only occasionally suitable as therapeutic proteins (Knight et al., 1995).

Clearly this approach is not amenable to the high throughput screening of antibody libraries since with the current technology it is not possible to refold millions or tens of millions of antibodies individually.

A further problem is that since E. coli expressed antibodies are not glycosylated, they fail to bind to complement factor 1q (C1q) or Fc and many other Fc receptors.

Nonetheless, since the aglycosylated antibodies fail to elicit complement activation and can not mediate the recruitment of immune cells such as macrophages, they have previously been ineffective for many therapeutic applications.

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